Live attenuated influenza vaccine

ABSTRACT

The invention relates to a simple and efficient process for isolating viruses from various sources and for producing live attenuated influenza vaccines in a serum-free Vero cell culture under conditions where alterations in the surface antigens of the virus due to adpative selection are minimized or prevented. The process does not require purification of the virus-containing supernatant harvested from the cell culture nor post-incubation treatment of the viruses for HA activation. The invention further relates to influenza A and B master strain candidates and to vaccines made thereof.

TECHNICAL FIELD

The present invention is in the field of virology and vaccinedevelopment and relates to an improved method of manufacture of a viralvaccine, particularly of a whole-virus vaccine, preferably of anattenuated live vaccine and to vaccines obtainable by the method.

BACKGROUND OF THE INVENTION

The influenza hemagglutinin (HA) antigen is the major target for theprotective immune responses of a host to the virus.

A common practice of recovering new viral isolates involves recoveryfrom a nasal or throat swab or from a similar source, followed bycultivation of the isolates in embryonated chicken eggs. The virusadapts to its egg host and large scale production of the virus can becarried out in eggs. Such conventional methodology involving embryonatedchicken eggs to produce Influenza vaccine is, however, extremelycumbersome, involving the handling of many thousands of eggs per week aswell as extensive purification of the virus suspension derived from theallantoic fluid to ensure freedom from egg protein.

Another disadvantage in the use of chicken embryos for virus productionlies in the fact that this substrate strongly favors the selection ofvirus variants that differ in their antigenic specificity from thewildtype virus and not rarely results in viruses that may not besuitable for vaccine production due to their altered phenotypesincluding, for instance, considerable reduction in immunogenicity.

Many attempts have therefore been undertaken in the art to utilizestandard tissue culture technology with established mammalian celllines, such as MDCK (Madin-Darby Canine Kidney) or Vero (African GreenMonkey Kidney) cells, for virus production, particularly influenza virusproduction.

One of the difficulties in growing influenza strains in tissue cellculture arises from the necessity for proteolytic cleavage of theinfluenza hemagglutinin in the host cell. Cleavage of the virus HAprecursor into the HA1 and HA2 subfragments, although not necessary forthe assembly of the viral elements to form a complete virion, isrequired, however, to render the virion infective, i.e. to enable it toinfect a new cell.

It has been reported. (e.g. Lazarowitz et al., “Enhancement of theInfectivity of Influenza and B Viruses by Proteolytic Cleavage of theHemagglutinin Polypeptide”, Virology, 68:440-454, 1975) that the limitedreplication of several influenza A strains in standard cell culturescould be overcome by the addition of proteases like trypsin to thetissue culture medium. Yet, there remained difficulties in some cases,for instance when using Vero cells.

Kaverian and Webster (J Virol 69/4:2700-2703, 1995) report that in Verocell cultures, and less pronounced in MDCK, swine kidney, or rhesusmonkey kidney cell cultures, the trypsin activity in the medium rapidlydecreased from the onset of incubation resulting in the failure of virusaccumulation in the medium due to the lack of production of a sufficientnumber of infective virions. They concluded that a trypsin inhibitingfactor was released from the Vero cells. They further showed that byrepeated addition of trypsin reproduction of virus could be resumed andmaintained for a number of reproduction cycles resulting in a muchbetter virus yield.

Another way for efficient vaccine production was reported in U.S. Pat.No. 5,753,489 wherein serum-free medium was used for virus propagationin a number of different mammalian cells including MDCK and Vero cells.The method disclosed therein comprises growing vertebrate cells inserum-free medium, infecting the cell culture with a virus, incubatingthe cell culture infected with the virus, removing a portion of thevirus-containing medium and contacting this portion. with a protease,thereafter adding to that portion a protease inhibitor and returningthat portion to the cell culture. It is preferred therein to provide thesteps of growing, infecting and incubating in a first vessel and thesteps of trypsin-contacting and inhibitor-adding are performed in asecond vessel connected with the first vessel in a loop so that thesteps o can be performed in a closed cycle. This system allows to usetrypsin or other proteolytic enzymes at much higher concentrations thanthose normally tolerated by cells in culture.

EP 0870508 reports a method to produce a viral antigen vaccinecomprising infecting an animal cell line, optionally a Vero cell line,with virus, propagating virus in the cell culture, adding a nucleaseenzyme to the cell culture shortly before the end of virus propagationto digest nucleic acid material released from the lysing host cells intothe medium, harvesting the virus and obtaining viral antigens thereof byextraction in order to make the viral antigen vaccine. The patent issilent with regard to the kind of nutrient medium used for viruspropagation and also with regard to the addition of a protease, usuallyrequired for the final processing of influenza virus hemagglutinin toget infectious virus. The method further requires various purificationsteps for providing a ready-for-use vaccine preparation.

It is known, however, that the nature the host substrate as well as thecomposition of the nutrient medium used for virus propagation maysignificantly affect immunogenicity and antigenicity of the virusprogeny obtained therewith. Particularly, serum-containing media may notonly decrease antigenicity of viral progeny but additionally maydecrease protease activity in the medium, hence inhibit virusmaturation, and subsequently require expensive steps of purification.

SUMMARY OF THE INVENTION

The present invention overcomes the drawbacks of the prior art. Itrelates to a simple and efficient process for isolating viruses fromvarious sources and for producing viral progeny for use as vaccines,particularly live attenuated influenza vaccines, in under conditionswhere alterations in the surface antigens of the virus due to adaptiveselection are minimized or entirely prevented.

It is also an object of the present invention to provide for a methodfor the production of viruses, particularly influenza viruses, thatyields viral progeny that selectively agglutinates human erythrocytesbut not chicken erythrocytes, and that preferably has antigenicproperties identical with those of the initially inoculated virusstrain, e.g. a primary clinical wildtype isolate.

In a preferred embodiment, the nucleic acid sequence of the HA gene andoptionally of the NA gene of the propagated virus is identical with theone of the initially inoculated strain (e.g. an epidemic strain, primaryclinical isolate of an infected patient).

It is another object of the invention to provide a method for efficientproduction of a whole-virus vaccine, particularly a live attenuatedvaccine, in a single step procedure that does not require anychromatographic or other purification steps of the virus suspensionharvested from the cell culture supernatant by centrifugation,particularly no protein separation or purification steps.

It is yet another object of the invention to provide attenuated, coldadapted and temperature sensitive influenza A and B strains and vaccinesmade thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic illustration of the time course of trypsininactivation in the supernatant of a Vero cell culture.

FIG. 2 is a graphic illustration of the time course of trypsininactivation in the supernatant of a MDCK cell culture.

DETAILED DESCRIPTION OF THE INVENTION

Comparative experiments using embryonated eggs, MDCK and Vero cellsclearly proved that the initially inoculated virus is likely to undergoeantigenic alteration during growth on any one of these substrates

Our experiments confirmed that the alterations are least or even absentfor influenza virus strains grown on Vero cells in serum-free medium.Moreover, it turned out that influenza A viruses, at least strains ofthe H3N2 subtype, when multiplied on Vero cells in serum-free andprotein-free medium exhibit a selectivity for agglutination of humanerythrocytes but not for chicken erythrocytes. Also, they did not growon eggs. This was a first indication that these Vero-grown viruses mightbe more identical with the wildtype virus of the corresponding clinicalisolate than the ones grown on MDCK cells or eggs.

Indeed, comparison of the HA and NA gene sequences of wildtype isolatesobtained from nasal swabs with the ones of the same viruses after growthon Vero and MDCK cells, respectively, revealed alterations in the HA orNA of MDCK-grown viruses relative to the HA or NA of the swab isolatesor of the Vero-grown viruses or of both the swab isolates and theVero-grown viruses.

Moreover, experimental data obtained from immunizations of ferrets withVero- and MDCK-grown wildtype viruses indicate a far stronger virulenceof the Vero-grown viruses compared to the MDCK-grown viruses. Also, theimmunogenicity of the Vero-grown viruses tested in an animal trial onmacaques was demonstrated to be significantly superior to the one of theviruses grown on MDCK cells or eggs.

These findings together provide strong evidence for the hypothesis thatthe process for the multiplication and propagation of viruses accordingto the present invention as hereinafter described in more detail yieldsviruses that are either unaltered compared to the initially inoculated(e.g. wildtype) virus or are modified to only a minor extent.

It is not only the avoidance of antigenic alterations that makes thepresent process of virus multiplication so unique, but it is also itsstriking simplicity which makes it extremely suitable for large scaleindustrial vaccine production.

Further experiments have shown that the source of trypsin (ortrypsinogen) may be one additional factor that influences the overallyield of infective virions. Indeed, while the methods known in the art(e.g, Kaverin and Webster, J Virol 69/4:2700-2703, 1995; or U.S. Pat.No. 5,753,489) use either repeated addition of trypsin (Kaverin andWebster) or high trypsin concentrations (U.S. Pat. No. 5,753,489), theprocess according to the present invention applies only half or less ofthe trypsin concentrations reported in the prior art. Moreover, a singleaddition of as little as 0.5-10 μg, preferably 2-5 μg trypsin per ml tothe cell culture medium prior to or at the beginning of incubation ofthe infected host cells is sufficient to reach optimal infective virustiters. Inactivation experiments revealed that porcine or humanrecombinant trypsins are far less susceptible to inactivation by Vero orMDCK cells than bovine trypsin. Since bovine trypsin is most commonlyused in the art it is rather likely that prior art literature unlessexplicitly mentioning another trypsin source, implicitly refers tobovine trypsin only. This would also help to explain the modes andconcentrations of trypsin application recited, for instance, in Kaverinet al. and in U.S. Pat. No. 5,753,489.

Using porcine or human rec trypsin or trypsinogen for initiallysupplementing the serum-free medium for Vero cell cultures according tothe present invention therefore allows to use extremely low trypsin ortrypsinogen concentrations and thus prevents the need of labor-intensiveand costly purification steps after harvesting of the virus-containingsupernatant.

Another step that contributes to make the present process simple andtherefore attractive to vaccine manufacturers is the addition of asingle dose of highly active endonuclease to the cell culture mediumprior to or at the beginning of incubation of the infected Vero cellsfor virus propagation. This endonuclease, preferably Benzonase™, isadded once to the medium at a very low initial concentration of 2-30,preferably 5-15, Units per ml of medium and effectively clears the cellculture medium from free DNA and RNA originating mainly from the lysingor lysed host cells. The residual Benzonase enzyme concentration in theready-for-use vaccine preparations obtained from the centrifugedsupernatant remains at 5 ng or less per dose.

Benzonase™ is a trademark of Nycomed Pharma A/S Denmark and relates toan extracellular unspecific endonuciease obtained from Serratiamarcescens. Benzonase is a genetically engineered endonuclease whichdegrades both DNA and RNA strands in many forms to smalloligonucleotides. It promotes quick reduction of the viscosity of celllysates, which facilitates ultracentrifugation. It reduces proteolysisand increases the yield in targeted protein and offers completeelimination of nucleic acids from, e.g. recombinant, proteins. It has anexceptionally high activity of 400,000 U/mg.

A third and important advantage of the present process is the factortime hence process costs. Due to the use of serum-free medium that doesnot contain proteins of animal origin and preferably no antibiotics,expensive and time-consuming purification procedures can be reduced to aminimum or even totally avoided. Also, because the addition of exogenousenzymes such as the protease (e.g. trypsin or trypsinogen) and nuclease(e.g. Benzonase) occurs once at the beginning of the virus propagationphase this saves plenty of time that the state-of-the-art methodsrequire for post-incubation treatment of the virus-containing culturesupernatant (e.g., HA activation, RNA/DNA digestion, proteinpurification, etc.).

Surprisingly, it turned out that the early addition of either or both ofprotease (e.g. trypsin or trypsinogen) and nuclease (e.g.Benzonase) tothe virus-infected Vero-cell culture had no negative implications on thevirus yield, which is probably due to the very low enzyme concentrationsapplicable in the process of the present invention.

The present process of virus propagation is useful for themultiplication of various kinds of viruses, particularly influenza Aviruses of the H3N2 subtype, but is also suitable for the isolation andreproduction of any epidemic or laboratory influenza virus strain,regardless of the kind of virus inoculum (e.g., blood serum sample,nasal wash, nasal swab, pharyngeal swab, saliva, etc.). Using theprinciples of this process, a number of influenza A and B vaccines hasbeen produced which are part of the present invention and which arecharacterized in more detail in the subsequent Examples.

Also, protective efficacy as well as vaccine safety have been confirmedfor the vaccines made according to the present invention, as will bedemonstrated in the Examples.

The term “protein-free” or “free of non-serum proteins” as used hereinin connection with the method of virus multiplication or propagationaccording to the present invention shall mean free of any functionallyactive protein. It shall not exclude, however, non-functional peptidesas may originate from protein hydrolysates such as yeast extract or soyaextract. Unless stated otherwise, the term “protein-free”0 shall neitherexclude the presence of a protease and a nuclease enzyme at theconcentrations disclosed and claimed herein.

In a preferred embodiment, the present invention relates to a simple,reliable and highly economic method for the manufacture of a whole-virusvaccine, preferably of an attenuated live vaccine, comprising the stepsof:

-   a) infecting African Green Monkey Kidney (Vero) cells with a desired    virus, wherein the Vero cells have been grown in and separated from    a serum-free medium that is also free of non-serum proteins;-   b) combining the infected cells with a suitable serum-free cell    culture medium that is also free of non-serum proteins except for a    protease and a nuclease; and-   c) incubating the cells in the presence of said protease and said    nuclease to allow for production of infectious virus and,    simultaneously, for digestion of nucleic acid material released to    the cell culture medium;-   d) harvesting infectious virus by collecting virus-containing    supernatant obtained from centrifugation of the cell culture; and-   e) preparing a vaccine thereof comprising subjecting the    virus-containing supernatant to at least one processing step    selected from the group consisting of filtering, concentrating,    freezing, freeze-drying, and stabilizing by addition of a    stabilizing agent.

It is preferred that the virus used for propagation has never had anycontact to a host substrate other than a Vero cell line. This willensure best results with regard to immunogenic and antigenic identity ofthe initial virus (e.g. nasal swab isolate) and the viral progenyobtained after propagation.

It is also preferred that the virus used for propagation, particularlyfor the manufacture of a whole-virus vaccine, preferably an influenzaattenuated live vaccine, is an influenza virus selected from the groupconsisting of strains A/Sing/1/57ca, A/Sing/1/57ca/ΔNS 87,A/Sing/1/57ca/ΔNSPR8, A/Sing/1/57ca/NS124PR8, B/Vienna/1/99ca,B/Vienna/99/ca37 and any attenuated variants and reassortants derivedfrom any one of these strains. The genetic characteristics of thepreferred virus strains, e.g. master strains, are disclosed in fulldetail in the subsequent Examples.

Influenza strains A/Sing/1/57ca, A/Sing/1/57ca/ΔNS87,A/Sing/1/57ca/ΔNSPR8, A/Sing/1/57ca/ΔNS 124PR8, B/Vienna/1/99/22ca, andB/Vienna/1/99/37ca were deposited at the National Bank of IndustrialMicroorganisms and Cell Cultures located at 1113 Sofia, 125 Tsarigradskochausse blvd., bl. 2, P.O. Box 239, Bulgaria on May 9, 2006. The depositof A/Sing/1/57ca, with a scientific description, was assigned NBIMCCNumber 8460. The deposit of A/Sing/1/57ca/ΔNS87, with a scientificdescription, was assigned NBIMCC Number 8461. The deposit ofA/Sing/1/57ca/ΔNSPR8, with a scientific description, was assigned NBIMCCNumber 8462. The deposit of A/Sing/1/57ca/ΔNS124PR8, with a scientificdescription, was assigned NBIMCC Number 8463. The deposit ofB/Vienna/1/99/22ca, with a scientific description, was assigned NBIMCCNumber 8464. The deposit of B/Vienna/1/99/37ca, with a scientificdescription, was assigned NBIMCC Number 8465.

In another embodiment, the present invention refers to a whole-virusvaccine itself, preferably to an attenuated live vaccine, which in itsready-for-use form comprises essentially unmodified, optionally filteredand/or concentrated, virus-containing supernatant of a serum-free andprotein-free Vero cell culture used for production of said virus. It isparticularly preferred that the vaccine is produced according to themethod of the present invention as disclosed and claimed herein.

This “one-step” vaccine, which does not require further processing,e.g., purification steps other than centrifugation and/or conventionalfiltration (i.e. not gel filtration), is compliant with the requirementsfor FDA approval.

The term “essentially unmodified” as used herein with regard tovirus-containing supernatant in vaccine preparations according to thepresent invention shall refer to the composition of the supernatant asis at the time of harvesting the propagated virus, i.e. to thecomposition of the soluble components and ingredients present in theliquid phase of the supernatant. Minor alterations of the composition ofingredients as may occur due to steps of, for example, filtration,sterile filtration, centrifugation, concentration, drying, orfreeze-drying of the virus-containing supernatant, shall be regarded asfalling within the scope of “essentially unmodified”. Also, the termshall not exclude the presence of preserving and/or stabilizing agentsusually applied in the art to vaccine preparations.

The whole-virus vaccines of the present invention may be used for theprophylactic or therapeutic treatment of viral infections, particularlyof influenza virus infections. They may be administered as known in theart, e.g. intravenously, subcutaneously, intramuscularly or, mostpreferably, intranasally. The virus strains disclosed herein and thevaccines made thereof may, however, also be used as vectors or shuttlesto present heterologous antigens to the immune system, e.g. antigens ofviral envelope proteins such HIV-1 or hepatitis antigens.

Further preferred embodiments are defined in the dependent claims.

In order that the invention described herein may be more fullyunderstood, the following Examples are set forth. They are forillustrative purposes only and are not to be construed as limiting thisinvention in any respect.

EXAMPLE 1 Virus Production

Cultivation of Vero/SF (=serum-free) cells:

-   SF-Medium: DMEM (Biochrom F0435), Ham's F12 (Biochrom F0815), 5 mM    L-Gln, 0.1% SF-supplement (a) or (b); antibiotics (only for first    passage of virus isolation).-   SF-Supplement: protein hydrolysate of non-animal origin, without    functional proteins such as insulin, transferrin or growth factors:    -   a) 62.5 g hy-soy/UF, Quest 5X59100, to 500 g HQ-water, filtered        with PES 0.2 μm filter;    -   b) 12.5 g hy-pep 1510, Quest, to 100 g HQ-water, filtered with        PES 0.2 μm filter.

The content of a deep frozen (liquid nitrogen) disinfected (70% ethanol)ampule of WCB Vero cells was thawed and added to 9 ml of cold serum-free(SF) medium in a 10 ml tube and centrifuged for 10 min at 1000 rpm (170g). The pellet was resuspended in SF-medium to a total of 30 ml,transferred to a 80 cm² Roux bottle and incubated at 37° C. and 7% CO₂for at least 15 min. Thereafter, the medium was removed and the cellswere washed with approx. 0.1 ml/cm² PBS def.(=PBS without Ca²⁺ andMg²⁺). Addition of trypsin/EDTA-solution (8-10 μl/cm²; 0.1%trypsin/0.02% EDTA-solution) and incubation at room temperature forabout 3 min. Detaching by gently pushing the Roux bottle against palm ofthe hand, addition of SF-medium and trypsin inhibitor (Sigma, T6522) ata quantity of about ⅕ of volume of the trypsin/EDTA solution.Repartition of the cell suspension to Roux bottles or roller bottles,incubation at 37° C. and 9% CO₂.

MDCK cells were grown in DMEM/Ham's F12+2% FCS (heat inactivated);embryonated hen eggs were 11-12 days old and of SPF (specific pathogenfree) origin.

Propagation of Virus Strains:

Old medium from roller bottles containing Vero cells was removed andcells were infected with virus by addition of 5 ml virus suspension inSF-medium to each roller bottle, resulting in an MOI (multiplicity ofinfection) of approximately 0.01. After incubation for 45 minutes at 33°C. the virus inoculum was removed with a pipette. 90 ml of SF-mediumsupplemented with 0.5-10, preferably 2-5 and most preferably 2 μg/mlporcine trypsin (supplier: AvP) or human recombinant trypsin ortrypsinogen (own production) and 0.5 g/l sodium bicarbonate were addedto each roller bottle and the bottles incubated at 33° C. and 5% CO₂.For the production of attenuated live vaccine samples for use in animaltesting and in human clinical trials the SF-medium was supplemented withtrypsin and, additionally, with Benzonase™ at a concentration of 2-30,preferably 5-15, and most preferably 10 Units of Benzonase™ per ml ofmedium. Virus was harvested after 64 hours post infection bycentrifugation of the culture supernatant for 5 min at 4000 rpm (3000 g)at 10° C. in 50 ml-tubes. The supernatant was pooled for each virusstrain and stored at +4° C. Aliquots thereof were used for vaccinetesting.

For storage purposes the virus preparations may be freeze-dried andstabilizer such as, for example, trehalose and lactalbumin enzymatichydrolysate in HEPES buffer may be added. Reconstitution may be donewith sterile water.

EXAMPLE 2 Comparison of Trypsin Inactivation in Cell Cultures

TABLE 1 Trypsin inactivation in Vero vs. MDCK cell culture Vero/MDCK 0 h24 h 48 h 72 h bovine trypsin 0.314/0.314 0.199/0.239 0.110/0.2010.026/0.203 porcine trypsin 0.230/0.230 0.201/0.206 0.171/0.2090.133/0.201 (high) porcine trypsin 0.129/0.129 0.108/0.118 0.081/0.0990.054/0.116 (low) human rec 0.097/0.097 0.054/0.088 0.026/0.0800.008/0.076 trypsin

Supernatants obtained from uninfected Vero cell cultures (grown in SFmedium as described in Example 1) and MDCK cell cultures (grown inFCS-supplemented medium as described in Example 1) were tested for theircapacity to inactivate trypsin of different origin that has been addedto the supernatant at time =0 h at equal concentrations each. Porcinetrypsin has been applied in two different qualities (obtained fromdifferent manufacturers), i.e. with high or low activity. The resultsare presented in Table 1 and in FIGS. 1 and 2.

The data unambiguously show that bovine trypsin is rapdily inactivatedin Vero cell culture supernatant and less rapidly in MDCK cell culturesupernatant. Porcine and human rec trypsin (manufactured in ourlaboratories) remain fully active in MDCK supernatants while they aregradually inactivated in Vero supernatants at approximately half or lessof the velocity of bovine trypsin inactivation. The difference of theporcine trypsins tested is only in the starting OD-level at 247 nm,while the inactivation characteristics are essentially identical forboth lots of porcine trypsin.

EXAMPLE 3 Comparison of Various Viral Properties After Growth onDifferent Host Cell Substrates

Virus propagation was carried out as described in Example 1 for thedifferent host cell substrates. Each of the seven isolates recovered onVero cells was reactive with human erythrocytes but not with chickenerythrocytes and none of them accumulated in embryonated eggs. On theother hand, all isolates recovered on MDCK cells were reactive both withchicken and human erythrocytes and were capable of growing in eggs.Although these differences were not seen in influenza A viruses of theH1N1 substype nor in influenza B isolates (see subsequent Tables 3 and4), it may nevertheless be assumed that cultivation of influenza viruseson Vero cells will maintain antigenic properties more properly thancultivation on other substrates.

TABLE 2 Characteristics of H3N2 viruses isolated from clinical materialon Vero/SF cells HA titer with Isolate Antigenically Isolated chickenhuman Growth in number related to on erys erys eggs A/47/96A/Johannesburg/ Vero − + − 33/94 MDCK + + + A/7729/98 A/Sydney/5/97 Vero− + − MDCK + + + A/1143/99 A/Sydney/5/97 Vero − + − MDCK + + + A/1144/99A/Sydney/5/97 Vero − + − MDCK + + + A/1179/99 A/Sydney/5/97 Vero − + −MDCK + + + A/1180/99 A/Sydney/5/97 Vero − + − MDCK + + + A/1182/99A/Sydney/5/97 Vero − + − MDCK + + +From the data in Table 3 it appears that H1N1 influenza viruses may beless susceptible to adaptive selection, as the Vero and MDCK-grownisolates do not exhibit significant differences in theirhemagglutination characteristics nor in their HA sequences. A similarconclusion may be drawn for the B isolates listed in Table 4.

The clinical starting material (e.g. serum samples, swabs) for virusisolation and replication was primarily obtained from:

-   1. Institute of Virology, Vienna, Austria (Prof. F. Heinz) 1995/96,    1996/97-   2. Unité de Génétique Moléculaire des Virus Respiratoires, Institute    Pasteur, Paris, France (Prof. S. van der Werf) 1996/97-   3. Public Health Laboratory Service, London, UK (Dr. M. Zambon)    1996/97-   4. Laboratoire Central de Virologie, Hôpitaux Universitaires de    Genéve, Geneva, Switzerland (Dr. W. Wunderli) 1996/97, 1997/98-   5. Virus Unit, Queen Mary Hospital, Hong Kong (Dr. W. L. Lim)    1997/98

TABLE 3 Characteristics of H1N1 viruses isolated from clinical materialon Vero/SF cells Changes HA titer with in HA1 at Isolate Antigenicallychicken human position number related to Isolated on erys erys Growth ineggs 225 A/5389/95 A/Bayern/7/95 Vero + + + D MDCK + + + D A/1035/98A/Beijing/262/95 Vero + + + D MDCK + + + D Egg + + + G Swab D A/1131/98A/Beijing/262/95 Vero + + + D MDCK + + + D Swab D A/1134/98A/Beijing/262/95 Vero + + + D MDCK + + + D Egg + + + n.t. Swab D

TABLE 4 Characteristics of B viruses isolated from clinical material onVero/SF cells Changes HA titer with in HA1 at Isolate Antigenicallychicken human position number related to Isolated on erys erys Growth ineggs 198 B/4291/97 B/Beijing/184/93 Vero + + + identical MDCK + + +B/1/99 B/Beijing/184/93 Vero + + + T(g.s) MDCK + + + T(g.s) EGG + + + ASwab T(g.s) B/110/99 n.t. Vero + + + identical MDCK + + + B/147/99 n.t.Vero + + + identical MDCK + + + B/156/99 B/Beijing/184/93 Vero + + +identical MDCK + + + B/157/99 B/Beijing/184/93 Vero + + + identicalMDCK + + +

TABLE 5 Amino acid changes in HA, NA and M proteins of H3N2 influenzaviruses isolated on different host systems Changes at positions HA NAIsolate number 128 129 229 133 218 220 136 151 M A/47/96 Vero T(g.s)A/47/96 MDCK A A/7729/98 Vero E R A/7729/98 MDCK G K A/1143/99 SwabN(g.s) G n.t n.t n.t A/1143/99 Vero N(g.s) G D identical A/1143/99 MDCKD E G A/1144/99 Swab R n.t n.t A/1144/99 Vero R identical identicalA/1144/99 MDCK G A/1179/99 Swab identical n.t n.t A/1179/99 Veroidentical identical A/1179/99 MDCK A/1180/99 Swab identical n.t n.t n.tA/1180/99 Vero Q identical A/1180/99 MDCK R A/1182/99 Swab identical n.tn.t A/1182/99 Vero n.t n.t A/1182/99 MDCK n.t n.t

The results show that with some isolates there was no alteration of theHA sequence of Vero or MDCK propagated viruses over the HA sequencedirectly obtained from the swab material by PCR amplification. In someother isolates grown on MDCK cells the HA and/or NA sequences weredeviating from the corresponding sequences obtained on Vero cells. TheVero-derived viruses did not show, however, any deviations in the HAsequence over the HA sequence of the swab isolates, where determined.

TABLE 6 Immunogenicity of Vero-, MDCK- and Egg-derived viruses formacaques Animal Virus for Dose, number immunization PFU/ml Serum HItiters 96 A/Vienna/47/96 V 5 × 10⁴ 256 88 A/Vienna/47/96 V 5 × 10⁴ 12815 A/Vienna/47/96 V 1.0 × 10⁶   128 95 A/Vienna/47/96 V 1.0 × 10⁶   25693 A/Vienna/47/96 M 1.0 × 10⁶   16 128 A/Johannesburg/33/94 E 5 × 10⁶ 32110 A/Vienna/157/97 V 5 × 10⁴ 128 78 A/Wuhan/359/95 E 5 × 10⁶ 32 TheMacaques were immunized i.n. in the absence of anesthesia with 1 ml ofvirus suspension V—Vero- isolated virus M—MDCK- isolated viruses E—eggisolated viruses

TABLE 7 Virulence of Vero- and MDCK- derived variants of A/Vienna/47/96wt virus for ferrets Number of Virus animals with dose, fever on dayViruses PFU/ml 1 2 3 A/Vienna/47/96 Vero 2 × 10² FF FFF 1 × 10³ FFF FFFA/Vienna/47/96 MDCK 5 × 10² 5 × 10³ FF 5 × 10⁴ FF F F Animals wereimmunized i.n. under ether narcosis with 1 ml of virus suspension.N—normal temperature from 38.1° C. to 39.9° C.; F—fever, more than 40.0°C.

The most surprising, yet important result in Table 6 is the very lowimmunogenicity of MDCK-derived A/Vienna/47/96 virus compared with thecorresponding Vero-derived virus. It is no particular surprise that theegg-derived viruses show only poor immunogenicity.

Similarly, the results listed in Table 7 indicate that Vero-derivedviruses are less, if at all, altered by adaptive selection on their hostsubstrate in comparison to MDCK-derived viruses. This means thatrelative to the MDCK-derived viruses the Vero-derived viruses maintainmore or even all of the immunologically relevant, particularlyantigenic, properties of the original virus.

EXAMPLE 4 Vaccine Production with Preferred Strains

The process described in Example 1 was also used for the production ofvaccine samples for animal testing and human clinical studies. It isunderstood that the process of virus propagation described therein alsoencompasses variations that could be suggested or applied by a person ofordinary skills in the art without inventive input and as long as thevariations do not change the sense of the present invention as describedherein and in the claims.

Vaccine samples containing one or more of the preferred influenza A or Bwildtype strains, master strains or reassortant strains (that aresubsequently described in more detail) were exclusively produced usingthe continuous Vero cell line as the host cell system (unless forpurposes of comparison with samples obtained from other host substrates)in serum-free medium additionally supplemented with the nutritionalingredients and enzymes as described in Example 1.

Some methods suitable for modifying wildtype viruses including themethods of attenuation (e.g., temperature sensitivity), cold adaptationand reassortment are known in the art and extensively reviewed, forinstance, in WO 99/64068.

Further characteristics of the two most preferred influenza A and Bmaster strain candidates useful for attenuated live vaccine production,e.g., by 6/2 reassortment with the HA and NA genes of actual epidemicinfluenza viruses recommended by the WHO, are given in the followingTables 8-13.

TABLE 8 Characteristics of master strain candidates for live influenzavaccines Influenza A A/Singapore/1/57/ca Influenza B H2N2B/Vienna/1/99/ca Passage A/Singapore/1/57 wt B/Vienna/1/99 wt historyegg derived H2N2 Vero derived 20 passages at 37° C. on 1 additionalpassage at 33° C. Vero/SF cells on Vero/SF cells 25 passages at 25° C.on 22 passages at 25° C. Vero/SF cells on Vero/SF cells Method of Serialpassages at optimal Serial passages at optimal and attenuation andsuboptimal temperature suboptimal temperature on on heterologous systemheterologous system Phenotypic temperature sensitive (ts) temperaturesensitive (ts) markers cold adapted (ca) cold adapted (ca) very lowreproduction in very low reproduction in mouse lungs mouse lungsGenotypic Mutations: 13 (8 coding) Mutations: 5 (3 coding) markers PB2 3(2 coding) PB2 0 PB1 2 (1 coding) PB1 1 PA 4 (3 coding) PA 0 NP 1 NP 2(1 coding) M 2 (2 coding) M 1 NS 1 NS 1

TABLE 9 Full Sequence of the 8 genome segments and of the 10corresponding proteins of strain A/Singapore/1/57/ca A/Singapore/1/57/ca(H2N2) RNA Nucleotide sequence segment (cDNA) Protein Amino acidsequence 1 SEQ ID No. 1 PB2 SEQ ID No. 9 2 SEQ ID No. 2 PB1 SEQ ID No.10 3 SEQ ID No. 3 PA SEQ ID No. 11 4 SEQ ID No. 4 HA SEQ ID No. 12 5 SEQID No. 5 NP SEQ ID No. 13 6 SEQ ID No. 6 NA SEQ ID No. 14 7 SEQ ID No. 7M1 SEQ ID No. 15 M2 SEQ ID No. 16 8 SEQ ID No. 8 NS1 SEQ ID No. 17 NS2SEQ ID No. 18 ca—cold adapted

It shall be noted, however, that the genome segments No. 4 and 6, i.e.,the HA and NA genes, are not required to characterize the influenza Amaster strain candidates, because these genes will be exchanged for thecorresponding genes of actual epidemic influenza viruses (as mentionedhereinbefore). The features important for the safety of a vaccine, e.g.temperature sensitivity, or features that allow intranasaladministration of a vaccine, namely cold adaptation (because the averagetemperature in a nose is lower than the usual body temperature), areprimarily caused by mutations in the remaining 6 genome segments.

The following Table 10 lists the mutations in the genome segments ofA/Singapore/1/57/ca compared to the corresponding wildtype strainA/Singapore/1/57/wt.

TABLE 10 Mutations in the genome segments of attenuated, temperaturesensitive, cold adapted influenza strain A/Singapore/1/57/ca compared toA/Singapore/1/57/wt strain RNA Nucleotides Amino acids seg- Lengthchanged Length changed ment (n″ds) position wt ca Protein (aa) positionwt ca 1 2341  252 a g PB2 771 — — —  581* t c 185 I T 1046* g t 340 R I2 2341 1279* t a PB1 757 419 L I 1965 a c — — — 3 2233  707* a t PA 716228 I N 1425 t a — — — 1537* a g 505 V I 1819* g c 598 Q E 5 1565  210 ga NP 506 — — — 7 1027  327* g a M1 252 101 R K  499* g c 158 Q R M2 97 —— — 8 890  813 a g NS1 237 — — — NS2 121 — — — Total number ofmutations - 13 (8 coding) *coding mutations

Preferred variants of A/Sing1/57/ca comprise the ones listed in thefollowing Table 11, wherein “Δ” means “del” or “delta” and stands for amutant that contains at least one “deletion” in its NS gene segment.

TABLE 11 Preferred variants of A/Sing/1/57/ca A/Sing/1/57/ca Sing ca/ΔNS87 Sing ca/ΔNSPR8 Sing ca/NS124PR8 PB2(Sing ca*)

PB1(Sing ca*)

PA(Sing ca*)

HA

NP(Sing ca*)

NA

M1, 2(Sing ca*)

NS1, 2(Sing ca*)

NS1, 2(PR8**)

Phenotypes ca + + + + ts + + + + IFN-induct. − +/− + + IFN-sensit − + +− *genome segment originating from A/Singapore/1/57/ca **genome segmentoriginating from influnza A/PR8/34 ca—cold adapted; ts—temperaturesensitive; aa—amino acid(s) IFN-induct.—strain causes interferon releasein host substrates that are able of IFN production, as well as in animalor human immune systems upon administration. IFN-sensit.—strain issensitive towards interferon; replication in IFN producing systems isreduced or stopped. Sing ca/ΔNS 87—strain A/Singapore/1/57/ca containingdeletion of 87 amino acids in NS1 gene at aa position 36-123. Singca/ΔNSPR8—strain A/Singapore/1/57/ca containing the NS gene segment fromA/PR8/34 (herein also abbreviated “PR8”) which contains a deletion ofthe entire NS1 gene. Sing ca/NS124PR8—strain A/Singapore/1/57/cacontaining the NS gene segment from A/PR8/34 which contains a stop codonat aa position 124 of the NS1 gene. +/− means that the phenotype needsfurther clarification and can not yet be unambiguously defined.

The following Tables 12, 13 and 13A refer to preferred influenza Bmaster strain candidates and to variations and reassortants,respectively, thereof.

TABLE 12 Full Sequence of the 8 genome segments and of the 11corresponding proteins of strain B/Vienna/1/99/ca B/Vienna/1/99/caNucleotide sequence RNA segment (cDNA) Protein Amino acid sequence 1 SEQID No. 19 PB2 SEQ ID No. 27 2 SEQ ID No. 20 PB1 SEQ ID No. 28 3 SEQ IDNo. 21 PA SEQ ID No. 29 4 SEQ ID No. 22 HA₀ SEQ ID No. 30 5 SEQ ID No.23 NP SEQ ID No. 31 6 SEQ ID No. 24 NB SEQ ID No. 32 NA SEQ ID No. 33 7SEQ ID No. 25 M1 SEQ ID No. 34 BM2 SEQ ID No. 35 8 SEQ ID No. 26 NS1 SEQID No. 36 NS2 SEQ ID No. 37 ca—cold adapted

The original strain B/Vienna/1/99 was isolated on Vero cell culturegrown with serum-free medium in February 1999 in Vienna, Austria from a12 year old female with acute influenza. It was rated asB/Beijing/184/93-like by the Center for Disease Control (CDC), Atlanta,USA. After an additional passage at 33° C. the wildtypestrain—designated as B/Vienna/1/99 wt—was attenuated by 22 serialpassages at 25° C. using the same cell culture system. The plaquepurification was done at 25° C. for the first and at 33° C. for thefollowing four rounds. The derived plaque purified clone was amplifiedand stored at −70° C., designated as B/Vienna/1/99 ca or briefly BV22.The identity as a B/Beijing/184/93-like virus was confirmed by HI-assaywith standard anti-serum from NIBSC.

TABLE 13 Mutations in B/Vienna/1/99/ca (=BV22) compared toB/Vienna/1/99/wt (BVie) 1. passage on Vero/SF Protein Segment (length in(lenght in Nucleotides changed amino Amino acids changed nucleotides)Position BVie BV22 acids) Position BVie BV22 1 (2396) — — — PB2 (770) —— — 2 (2369)  594 T C PB1 (752) — — — 3 (2305) — — — PA (726) — — — 4(1882)  457 G A HA₀ (584) 142 A T 1299 G T 422 K N 1595 G A 521 G E 5(1844)  128 C T NP (560)  23 S F  330 T C — — — 6 (1557) — — — NB (100)— — —  823 G A NA (466) 257 R Q 1135 T C 361 I T 7 (1190) — — — M1 (248)— — —  831 A G BM2  21 M V (109) 8 (1097)  116 G A NS1 (281)  25 A T — —— NS2 (122) — — —

TABLE 26 Characterization of B/Vienna/1/99 wt according to Los AlamosNational Library influenza database (db) (Web-adress: www.flu.lanl.gov)B/Vienna/1/99 Accession wt gene Nr. amino Accession Nr. coding for acidseq. nucleotide seq Remarks PB2, segment 1 ISDACH017 ISDNCHB017 in dblisted as segment 2 PB1, segment 2 ISDACH016 ISDNCHB016 in db listed assegment 1 PA, segment 3 ISDACH015 ISDNCHB015 HA, segment 4 ISDACH018ISDNCHB018 NP, segment 5 ISDACH013 ISDNCHB013 NA, segment 6 ISDACH012ISDNCHB012 M, segment 7 ISDACH011 ISDNCHB011 NS, segment 8 ISDACH014ISDNCHB014

In addition, further passaging of strain B/Vienna/1/99 ca for 15additional passages (i.e. a total of 37 passages on serum-free Vero cellculture) resulted in a mutant B/Vienna/1/99 ca37 (abbreviated BV37) withproperties even superior to the ones of BV22. This mutant contains anincreased number of mutations vis-à-vis BV22 and appears to be thecurrently most promising candidate for the production of a whole-virusvaccine, particularly for an attenuated influenza live vaccine, based ona non-recombinant influenza virus mutant. The additional mutations arelisted in Table 13A below:

TABLE 13 A Mutations for BV22 and BV37 compared to B/Vienna/1/99 wt 1stpassage on Vero/SF Segment Protein (length in Nucleotides changed(length in amino Amino acids changed nucleotides) Pos. BVie BV22 BV37acids) Pos. BVie BV22 BV37 1 (2396) — — — — PB2 (770) — — — — 2 (2369) 594 T C C PB1 (752) — — — — (BV37: 2370) 2348 — — A — — — — 3 (2305) —— — — PA (726) — — — — 4 (1882)  457 G A* A* HA₀ (584) 142 A T⁺ T⁺ 1122C C A 363 F F L 1299 G T G 422 K N K 1595 G A A 521 G E E 5 (1844)  128C T T NP (560)  23 S F F  212 C C T  51 P P L  330 T C^(#) C^(#) — — — —6 (1557) — — — — NB (100) — — — —  823 G A G NA (466) 257 R Q R 1135 TC^(•) C^(•) 361 I T^(•) T^(•) 7 (1190)  24 G G A M1 (248) — — — —  831 AG G — — — —  831 A G G BM2  21 M V V 1029 A A G (109)  87 I I V 8 (1097) 116 G A A NS1 (281)  25 A T T — — — — NS2 (122) — — — — Comparison withinfluenza sequence database 13.2.2001 (www.flu-lanl.gov): a) uniquemutations underlined in bold type; b) mutations common with: *B/Lee/40,B/Osaka/70, B/Kadoma/1076/99 (resulting amino acid: I) ⁺B/Lee/40,B/Osaka/70 ^(#)often: B/Lee/40, B/Ann Arbor/1/66 ca & wt,B/Singapore/222/79, B/North Dakota/83, B/Norway/1/84, B/Ibaraki/2/85,B/Ann Arbor/1/86, B/Victoria/2/87, B/Aichi/5/88 ^(•)B/Kanagawa/73

It shall be understood that the influenza A and B master strainsaccording to the present invention shall not be limited to the featuresand genetic characteristics explicitly listed in the tables herein butshall also comprise minor variations thereof as long as such variationsare in the sense of the present invention and do not subtantially alterany one of the functional features of the virus.

Such variations may occur, for instance, due to additional steps ofvirus multiplication or propagation (e.g. for the purpose of obtainingmaterial for sequence analyses).

Moreover, the gene sequences listed herein include the primer sequences(located at the beginning and at the end of each genome segment) thatwere used along with the present invention, which primer sequences maydiffer from the corresponding true sequences of the viral genomesegments of either or both the wildtype and the attenuated virusstrains.

EXAMPLE 5 Vaccine Safety and Efficacy

The subsequent data confirm temperature sensitivity and vaccine safetyfor influenza vaccines manufactured according to the present invention,e.g., as described in Example 1.

TABLE 14 Antibody response of mice after one intranasal immunisationwithout narcosis Number of Protection after Viruses responders¹ GMT³challenge² PR8/Sing ca - 2/6 0/6 <4 5/6 PR8/Sing ca - ΔNS 4/6 6.7 5/6PR8-wt 5/6 16.0 5/6 ¹number of animals with positive HI titer > 1:4²number of animals without detectable virus in the lungs ³Geometric meantiter of antibodies in serum PR8wt - influenza strain A/PR/8/34 wildtype(H1N1), pathogenic for mice PR8/Sing ca-2/6 - is the reassortant betweenattenuated influenza strain A/Sing/1/57 ca and PR8 wt, containing 2genes (HA and NA) from PR8wt virus and all other genes from A/Sing/1/57ca. PR8/Sing-ΔNS contains HA and NA genes from PR8wt, five genes fromA/Sing/1/57 ca and the NS gene of PR8 origin lacking the NS1 codingsequence (NS1 deletion or knockout).

TABLE 15 Antibody response and protection of mice after intranasalimmunisation with different variants of A/Singapore/1/57 virus (undernarcosis) Responders¹ 1-st 2-nd GMT after Protection immunisa- immunisa-two after Viruses tion tion immunisations challenge⁴ A/Sing/1/57/wt va²9/9  9/9 103.9 9/9  A/Sing/1/57/ca³ 8/10 10/10 55.7 8/10 A/Sing/57/ΔNS87 1/10 10/10 27.9 8/10 ¹number of animals with positive HI titer > 1:4²va—Vero-adapted ³ca—cold-adapted ⁴number of animals without detectablevirus in the lungs

TABLE 16 Reproduction of wt, va and ca variants of A/Singapore/1/57 inmouse lungs^(a) Virus titer in mouse lungs post infection on day,PFU/ml^(b) Viruses 2 4 6 A/Singapore/1/57/wt 1.6 × 10⁶ 2.2 × 10⁵ 1.4 ×10³ A/Singapore/1/57/wt va 2.5 × 10⁶ 2.1 × 10⁶ 1.0 × 10²A/Singapore/1/57/ca <10 <10 <10 ^(a)Mice were infected i.n. with 50 μlof virus fluid with a titer 1.0 × 10⁶ PFU/ml. ^(b)PFU/ml of 10% tissuesuspension, titrated on MDCK cells.

TABLE 17 Virulence of wt and ca variants of A/Singapore/1/57 virus forferrets Number of animals with fever post infection on day Viruses 1 2 3A/Singapore/1/57 wt FFF NNN NNN A/Singapore/1/57 ca NNN NNN NNN Rectaltemperature of animals was recorded twice a day and characterized asfollows: N—normal temperature from 38.1° C. to 39.9° C. F—fever, morethan 40.0° C. Each group consisted of 3 animals, which were immunizedi.n. under ether narcosis with 1 ml of virus fluid with a titer of 2 ×10⁶ PFU/ml.

TABLE 18 Reproduction of 2/6 reassortant of A/Hong Kong/1035/98 wt andA/Singapore/1/57/ca in mouse lungs^(a) Virus titer in mouse lungs on day2-6 post infection, PFU/ml^(b) Viruses 2 4 6 A/Hong Kong/1035/98 wt 6.8× 10⁴ 2.0 × 10⁴ <10 H1N1 A/Singapore/1/57/ca × <10 <10 <10 A/HongKong/1035/98 wt ^(a)Mice were infected i.n. under ether narcosis with 50μl of virus fluid. ^(b)PFU/ml of 10% tissue suspension, titrated onVero/SF cells, data are given as mean value for 6 mice (the lungs ofeach animal were treated separately). The reassortant contains the HAand NA genes from A/Hong Kong/1035/98 wt wildtype and the other 6 genesfrom A/Singapore/1/57/ca.

TABLE 19 Virulence of 6/2 reassortant of A/Vienna/47/96 wt andA/Singapore/1/57/ca for ferrets Virus Number of animals with fever onday Viruses subtype 1 2 3 Rhinitis^(b) Master strain A/Singapore/1/57/caH2N2 NNN NNN NNN ± Epidemic virus A/Vienna/47/96 wt H3N2 NNN FFF FFF +++Reassortant A/Singapore/1/57/ca × H3N2 NNN NNN NNN ± Vienna/47/96 wtAnimals were immunized i.n. under ether narcosis with 1 ml of virus, 2 ×10⁶ PFU/ml. N—normal temperature from 38:1° C. to 39.9° C.; F —fever,more than 40.0° C. ^(b)+++ - severe rhinitis ± absence of rhinitis

The results presented in Tables 16 to 19 clearly demonstrate the safetyof the vaccines containing the attenuated, temperature sensitive masterstrain or, in case of reassortants, of the vaccines based on thereassorted viruses composed of the “backbone” of the attenuated,temperature sensitive master strain (6 genes) and the HA and NA genesfrom, e.g., the pathogenic wildtype strain A/Hong Kong/1035/98 wt.

TABLE 20 Ts and ca phenotype of B/Vienna/1/99 PFU/ml on Vero cells atPFU/ml on MDCK cells at Virus 25° C. 33° C. 39° C. B/Vienna/1/99 wt <3004 × 10⁶ 4 × 10⁵ B/Vienna/1/99 ca (BV22) 1 × 10⁶ 2.4 × 10⁶   <20

TABLE 21 Genetic stability of the ts phenotype of B/Vienna/1/99 caPFU/ml on MDCK cells at Virus 33° C. 39° C. B/Vienna/1/99 wt 4 × 10⁶ 4 ×10⁵ B/Vienna/1/99 ca (BV22) 2.4 × 10⁶   <20 B/Vienna/1/99 ca (BV22) 8 ×10⁵ <20 after 5 passages at 33° C.

The strain BV22 was passaged five times at high MOI on Vero cells. Thenthe ts-phenotype was controlled again. The strain remained tmperaturesenssitive as can be seen in Table 21.

TABLE 22 Virulence of B/Vienna/1/99 ca and wt in mouse lungs PFU/ml* atday post infection Virus organ 2 3 4 B/Vienna/1/99 ca lung <20 <20 <20(BV22) nose   1 × 10²   1 × 10² 20 B/Vienna/1/99 wt lung   8 × 10⁴   7 ×10³ 4.4 × 10³ nose 3.8 × 10⁴ 3.4 × 10⁴ 1.4 × 10⁴ *9 OF1 mice per strainwere immunized intranasally under ether narcosis with 10⁵ PFU. At theindicated days post infection 3 mice per group were sacrificied. Lungsand nasal turbinates were homogenized for a 10% (w/v) suspension in PBSdef. A plaque assay of the suspensions was performed.

The data show that moderate reproduction of the ca master straincandidate BV22 was possible in the nasal mucosa while the ts property ofthe virus prevented reproduction in the lungs.

TABLE 23 Ts and ca phenotype of the reassortant influenza b strainPFU/ml on MDCK cells at Virus 33° C. 39° C. B/Vienna/1/99 wt 4 × 10⁶ 4 ×10⁵ B/USSR/69 wt 1.6 × 10⁶   4 × 10⁴ B/Vienna/1/99 ca (BV22) 1.4 × 10⁶  <20 BV22 × B/USSR/69 (6/2) 8 × 10⁶ <20

A 6/2 reassortant strain containing HA and NA of the wild type influenzastrain B/USSR/69 wt and the other 6 genome segments from B/Vienna/1/99ca (BV22) was established. The origin of the hemagglutinin was tested byHI-assay, all other genome segments by RT-PCT and restriction analysisusing methods known in the art.

TABLE 24 Virulence of the reassortant influenza B strain in mouse lungsPFU/ml* at day post infection Virus organ 2 3 4 B/Vienna/1/99 ca lung<20 <20 <20 (BV22) nose <20 1 × 10² 40 B/USSR/69 wt lung 1.8 × 10⁵ 4 ×10⁵ 2.4 × 10⁴ nose 1.6 × 10⁵ 2 × 10⁵ 1.6 × 10⁵ BV22 × B/USSR/69 wt lung<20 <20 <20 (6/2) nose 2.8 × 10³ 2 × 10³   4 × 10² *9 OF1 mice perstrain were immunized intranasally under ether narcosis with 10⁵ PFU. Atthe indicated days post infection 3 mice per group were sacrificied.Lungs and nasal turbinates were homogenized for a 10% (w/v) suspensionin PBS def. A plaque assay of the suspensions was performed.

EXAMPLE 6 Clinical Study

The following vaccines (in the form of nasal sprays) were producedaccording to the present invention (e.g. as described in Example 1) forintranasal delivery. Composition per ml (after reconstitution offreeze-dried material):

-   (1) Placebo: 2× SF-medium, 40 mM HEPES buffer, 8% lactalbumin    enzymatic hydrolysate, 4% trehalose;-   (2) Vero-Vac H1: A/Beijing/262/95 (H1N1)-like preparation comprising    4.3×10⁷ TCID₅₀ of 6/2 reassortant A/Singapore/1/57/ca with A/Hong    Kong/1035/98; 2× culture supernatant, 40 mM HEPES buffer, 8%    lactalbumin enzymatic hydrolysate, 4% trehalose;-   (3) Vero Vac H3: A/Sidney/5/97 (H3N2)-like preparation comprising    2.1×10⁷ TCID₅₀ of 6/2 reassortant A/Singapore/1/57/ca with    A/SW/7729/98; 2× culture supernatant, 40 mM HEPES buffer, 8%    lactalbumin enzymatic hydrolysate, 4% trehalose;-   (4) Russian trivalent vaccine (live influenza vaccine for adults):

A/17/Beijing/95/25 (H1N1) 1.1 × 10⁸ EID₅₀ A/17/Sidney/97/76 (H3N2) 2.3 ×10⁷ EID₅₀ B/60/Petersburg/95/20 1.1 × 10⁷ EID₅₀

-   (5) Monovalent Vero vaccine BV22: B/Beijing/184/93—like preparation    comprising 2×10⁶ TCID₅₀ of master strain candidate B/Vienna/1/99/ca    (=BV22); 2× culture supernatant, 40 mM HEPES buffer, 8% lactalbumin    enzymatic hydrolysate, 4% trehalose;

The vaccines were administrated to 13 volunteers per each vaccinationgroup. 550 μl of reconstituted vaccine (or placebo, respectively) weregiven intranasally to each patient on day 0 and for a second time on day22±1. The results are summarized in Table 25 below.

Safety Results:

The total number of adverse events (AE) during five days after the firstand second vaccination was 14 including 9 mild and 4 moderate AE. Onlyone volunteer showed severe AE, comprising an increase in bodytemperature up to 38.8° C. within 3 hours after the first vaccinationwithout any local or systemic symptoms. During the next four hours histemperature became normal again. After the first vaccinations 7 AE wereobserved, One of them was local and six were systemic. After the secondvaccination 2 local and 5 systemic AE were observed.

No significant difference in terms of safety was revealed between thegroups of the study including the one with placebo. No serious AErelated to the vaccination were observed except for the one mentionedabove. Two of the moderate AE occurred in the H3N2 group (temperatureelevation up to 37.6° and acute pharyngitis on day 3 in one volunteer;nasal obstruction, discomfort in the throat on day 22-24 and temperatureelevation up to 37.5° C. in another volunteer), and one in the H1N1group (pain in the throat, rhinitis from day 22-26, temperatureelevation up to 37-37.8° C. between days 22-24).

TABLE 25 Response of seronegative volunteers to Vero Vac vaccines and toa trivalent Russian cold-adapted egg derived vaccine % of volunteerswith at least 4-fold increase of serum HAI antibody titre Virus dose,TCID₅₀/ml or No. of to antigens No Vaccine for immunization EID₅₀/mlvolunteers H1N1 H3N2 B 1 Placebo 13 (8) 2 Vero Vac H1 (H1N1) 4.3 × 10⁷13 38 3 Vero Vac H3 (H3N2) 2.1 × 10⁷ 13 67 4 Russian trivalent vaccine:13 A/17/Beijing/95/25 H1N1 1.1 × 10⁸ 46 A/17/Sidney/97/76 H3N2 2.3 × 10⁷ 8 B/60/Petersburg/95/20 1.1 × 10⁷ 31 5 Vero vaccine BV22   2 × 10⁶ 1333 (8) patient developed spontaneous infection during course of study.

The results obtained from the clinical study thus confirm a very goodsafety of the vaccines produced according to the present invention andusing the preferred influenza A and B master strain candidates of thepresent invention.

1. A whole-virus vaccine in the form of an attenuated influenza livevaccine, comprising at least one influenza virus selected from the groupconsisting of strain A/Sing/1/57ca, strain A/Sing/1/57ca/ΔNS 87, strainA/Sing/1/57ca/ΔNSPR8, strain A/Sing/1/57ca/NS124PR8, strainB/Vienna/1/99ca, strain B/Vienna/1/99ca37, and a reassortant strain, thereassortant strain being composed of a backbone of five or six influenzagenome segments from one of the foregoing strains encoding proteinsother than HA and NA; and of a HA genome segment and a NA genome segmentfrom an influenza wildtype strain.
 2. The vaccine according to claim 1wherein it selectively agglutinates human erythrocytes but not chickenerythrocytes.
 3. The vaccine according to claim 1, wherein the backboneconsists of five of said genome segments.
 4. The vaccine according toclaim 1, wherein the backbone consists of six of said genome segments.